U.S. patent application number 11/435756 was filed with the patent office on 2006-09-21 for dna encoding anti-apoptotic protein and recombinant 30k protein.
This patent application is currently assigned to HANSON BIOTECH CO., LTD. of KOREA. Invention is credited to Eun Jeong Kim, Hye Jung Park, Tai Hyun Park.
Application Number | 20060210547 11/435756 |
Document ID | / |
Family ID | 35943759 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060210547 |
Kind Code |
A1 |
Park; Tai Hyun ; et
al. |
September 21, 2006 |
DNA encoding anti-apoptotic protein and recombinant 30K protein
Abstract
The present invention relates to DNAs encoding anti-apoptotic
30K proteins. More particularly, the present invention is directed
to 30K protein genes and a recombinant proteins prepared by using
novel anti-apoptotic gene obtained from silkworm. The present
invention also provides anti-apoptotic health care food,
pharmaceutical preparation, additive for cell culture medium, and
food supplement.
Inventors: |
Park; Tai Hyun; (Seoul,
KR) ; Kim; Eun Jeong; (Seoul, KR) ; Park; Hye
Jung; (Suwon, KR) |
Correspondence
Address: |
Gary M. Nath;NATH & ASSOCIATES PLLC
112 South West Street
Alexandria
VA
22314
US
|
Assignee: |
HANSON BIOTECH CO., LTD. of
KOREA
|
Family ID: |
35943759 |
Appl. No.: |
11/435756 |
Filed: |
May 18, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10926406 |
Aug 26, 2004 |
|
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11435756 |
May 18, 2006 |
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Current U.S.
Class: |
424/94.2 ;
435/184; 536/23.2 |
Current CPC
Class: |
C07K 14/43586
20130101 |
Class at
Publication: |
424/094.2 ;
435/184; 536/023.2 |
International
Class: |
A61K 38/54 20060101
A61K038/54; C07H 21/04 20060101 C07H021/04; C12N 9/99 20060101
C12N009/99 |
Claims
1-12. (canceled)
13. An anti-apoptotic additive for culture medium, comprising a
recombinant 30K protein.
14-19. (canceled)
Description
FIELD OF INVENTION
[0001] The present invention relates to DNAs encoding
anti-apoptotic protein and a recombinant 30K protein. More
particularly, the present invention is directed to novel
anti-apoptotic DNAs obtained from silkworm and a recombinant 30K
protein.
DESCRIPTION OF THE RELATED ART
[0002] Apoptosis is a normal physiologic process that leads to
individual cell death. This process of programmed cell death is
involved in a variety of normal and pathogenic biological events
and can be induced by a number of unrelated stimuli.
[0003] Changes in the biological regulation of apoptosis also occur
during aging and are responsible for many of the conditions and
diseases related to aging. Recent studies of apoptosis have implied
that a common metabolic pathway leading to cell death may be
initiated by a wide variety of signals, including hormones, serum
growth factor deprivation, chemotherapeutic agents, ionizing
radiation, and infection by human immunodeficiency virus
(HIV)(Wyllie (1980) Nature 284:555-556; Kanter et al. (1984)
Biochem. Biophys. Res. Commun. 118:392-399)
[0004] Apoptosis occurs sporadically in all tissues throughout life
and is a normal everyday occurrence; however, disproportionate
apoptosis, either excessive or deficient may cause serious
diseases.
[0005] Many researchers have found that several extant diseases
associated with apoptosis, particularly relates to cancer and
autoimmune disease which were caused by deficiency in apoptosis,
and dementia and Alsheimer's disease which were caused by surplus
apoptosis, and so called a degenerative disease and AIDS.
[0006] Subsquent researches have been developed for clinical trial
to treat above described by means of these anti-apoptotic gene and
proteins, or to use factors intervening signal transduction, that
induces apoptosis. These researches were concentrated on regulation
of apoptosis, induction of apoptosis, and biological mechanism.
[0007] Recently, it has been known to the public that several genes
such as Bcl-2 family inhibit the apoptosis effectively.
[0008] In actual, the study has been preceded to inhibit the
necrosis and apoptosis of PC12 cell induced by amyloid peptide
related to the demetia of the aged (Neurosci of Apoptosis
Protein:IAP). Also, to treat cancer, antisense technology of bcl-2,
hsp27 has been tried. Especially, treatment by anti-bcl-2 is known
to be effective in lymph tumor (J. Natl, Cancer Inst. 89,998(1997),
Lancet 349,1137(1997)).
[0009] By several result of study until now, as many regulative
factors of apoptosis have found, technology using regulator or
genome information of it has been developed from two points of
view.
[0010] One point is related to the study for treatment of the
disease induced by cell death. Second point is related to the study
of cell culture improving cell-production by inhibiting apoptosis.
These studies have been done by using bcl-2 family proteins and
genes.
[0011] The present inventors have conducted intensive researches in
regard to anti-apoptotic factor existing in silkworm hemolymph.
[0012] As a result, the present inventors have discovered a novel
method of fractionation, seperation, purificaton of proteins
obtained by silkworm hemolymph, which are capable to substitute for
conventional anti-apoptotic proteins encoded by genes such as
bcl-2, and that productivity of the recombinant protein and the
viability of host cells increase in insect/baculovirus system with
adding silkworm hemolymph to the culture medium (Biotechnol. Prog.,
15,1028(1999)).
[0013] Also, the present inventors have found that silkworm
hemolymph has inhibitory factor of apoptosis, and found that
silkworm hemolymph inhibits not only virus-induced apoptosis but
also apoptosis induced by various chemicals. Moreover, silkworm
hemolymph also inhibits human cell apoptosis.
[0014] The present inventor has also found these active factor is a
kind of protein, which is seperated from silkworm hemolymph (Korean
Patent Application No.10-2001-0010717, Biochem. Biophys. Res.
Commun., 285, 224(2001)). The corresponding genes are obtained by
Polymerase Chain Reaction (PCR) with the primers designed using
information of the above purificated protein, and the gene sequence
is analyzed.
[0015] The result of the gene sequence analysis indicated that the
gene is so called "30K protein" of which function had not yet been
known. The 30k protein group consists of five proteins and have the
sequence Id. No.1 to 5, respectively.
[0016] Also it had been known to the public that 30K proteins have
common characteristics in amino acid composition and immunological
activity as well as molecular weight and they are a group of
structurally related proteins with a molecular mass of
approximately 30,000 Da.
[0017] The genes encoding the 30K proteins were remarkably
different from the anti-apoptotic proteins such as bcl-2 family,
which had been known to the public.
[0018] The object of the present invention is to provide
anti-apoptotic protein originating from silkworm hemolymph. To
accomplish the object efficiently, recombinant DNA technology is
used to produce useful recombinant proteins in the present
invention.
[0019] The pET-22b(+) carrying the 30K protein gene is introduced
into E. coli BL(DE3). The 30K protein obtained from recombinant
cell is proven to have an effect on inhibiton of apoptosis.
[0020] According to the present invention, the anti-apoptotic
protein enables us to produce pharmaceuticals and health care food.
For example, the anti-apoptotic recombinant protein is effective to
dementia and Alsheimer's disease, which may be caused by surplus
apoptosis, and so called a degenerative disease and AIDS as
treatment. Also, the anti-apoptotic recombinant protein of the
present invention is applied to food additives and cell-culture
medium additives for improving productivity of incubating cells. On
the basis of the above discoveries, the inventors could have
accomplished the present invention.
[0021] Korean Patent Application No.10-2001-0010717,
No.10-2002-0059686 are referenced in this application in order to
more fully describe the state of the art to which this invention
pertains. The disclosure of each of these publications and patent
documents is incorporated by reference herein.
SUMMARY OF THE INVENTION
[0022] Therefore, the primary purpose of the present invention is
to provide DNA SEQ. ID. No. 1 encoding the 30Kc6, DNA SEQ. ID. No.
2 encoding the 30Kc12, DNA SEQ. ID. No. 3 encoding the 30Kc19, DNA
SEQ. ID. No. 4 encoding the 30Kc21 and DNA SEQ. ID. No. 5 encoding
the 30Kc23.
[0023] It is an another object of the present invention to provide
anti-apoptotic recombinant proteins comprising an amino acid
sequence set forth as SEQ. ID. No.6, ID. No.7, ID. No.8, ID. No.9
and ID. No.10.
[0024] It is a still another object of the present invention to
provide an anti-apoptotic pharmaceutical preparation comprising a
therapeutically effective amount of the recombinant anti-apoptotic
protein in a pharmaceutically acceptible carrier.
[0025] It is a yet another object of the present invention to
provide an anti-apoptotic health food, additive for culture medium
and food supplement comprising the recombinant protein.
[0026] It is a further object of the present invention to provide a
recombinant expression vector comprising the DNA of SEQ. ID. No.1
encoding anti-apoptotic 30Kc6 protein, ID. the DNA of SEQ. No.2
encoding anti-apoptotic 30Kc12 protein, the DNA of SEQ. ID. No.3
encoding anti-apoptotic 30Kc19 protein, ID. the DNA of SEQ. No.4
encoding anti-apoptotic 30Kc21 protein and ID. the DNA of SEQ. No.5
encoding anti-apoptotic 30Kc23 protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The above objects and other advantages of the present
invention will become more apparent by describing in detail a
preferred embodiment thereof with reference to the attached
drawings, in which:
[0028] FIG. 1A to FIG. 1C are the photographs of electrophoresis,
which indicate that the recombinant 30K protein expressed in E.
coli BL21(DE3)(A), BL21(DE3)(B) and purified recombinant 30K
protein(C).
[0029] FIG. 2A to FIG. 2B are the graphs which show the viability
of host cell measured 7 days after virus infection in the media
supplemented with the recombinant 30K protein described in the
above FIG. 1(c).
[0030] FIG. 3A to FIG. 3D are the FACS analytic chromatogram, which
show the effect of 30K protein on actinomycin D-induced insect cell
apoptosis. Except for (a), the cells were treated with 200 ng/ml
actinomycin D for 13 h and apoptosis was analyzed by flow
cytometry. (A)Sf9 cells not treated with actinomycin D, (B)Sf9
cells cultured in the medium containing 10% FBS, (C) Sf9 cells
cultured in the medium containing 5% FBS and 5% hemolymph, and
(D)Sf9 cells cultured in the medium containing 5% FBS and 0.2 mg/ml
recombinant 30K protein.
[0031] FIG. 4A(a) to FIG. 4A(C) are the photographs which show the
effect of 30K protein on staurosporine-induced human cell
apoptosis. HeLa cells were treated with 600 nM staurosporine for 12
h and apoptosis was analyzed by fluorescence microscopy using
Hoechst 33258 fluorescent dye. (A):(a) Cells cultured in the medium
containing 5% FBS and 5% hemolymph. (c) Cells cultured in the
medium containing 5% FBS and 0.2 mg/ml recombinant 30K protein.
[0032] FIG. 4B is the graph, which indicates percentage of
apoptotic cells represented in (B) was determined by counting the
number of apoptotic cells, which were detected by the method used
in (A).
DETAILED DESCRIPTION OF THE INVENTION
[0033] Cell death is categorized as either apoptotic or necrotic.
Apoptosis is a physiological cell death, which is morphologically
distinguishable from necrosis.
[0034] Necrotic cells are characterized by an overall increase in
size, mild clumping of chromatin and cell lysis.
[0035] However, apoptosis is different from necrosis where healthy
cells are destroyed by external processes, such as inflammation.
Apoptosis is a kind of voluntary, programmed death of cells that is
under genetic control. The cell's own genes play an active role in
its demise and is accompanied by the condensation of nuclei and
cytoplasm, the loss of microvilli, convolution of the plasma
membrane, and nuclear and cell segmentation.
[0036] Therefore, above objection of the present invention is
achieved by providing an anti-apoptotic recombinant anti-apoptotic
protein and DNAs encoding anti-apoptotic 30Kc6, 30Kc12, 30K19,
30K21, and 30K23 protein. It enables apoptosis to be inhibited
effectively in animal cells and human cells.
[0037] In one embodiment of the present invention, there is
provided anti-apoptotic protein synthesized by genetic
recombination technology using gene of protein separated from
silkworm homolymph.
[0038] DNAs of SEQ. ID. No.1 to 5 encoding anti-apoptotic 30Kc6,
30Kc12, 30K19, 30K21, and 30K23 protein, are obtained from
silkworm, repsectively.
[0039] A silkworm hemolymph has been used effectively in biological
researches. The production of recombinant protein in an insect cell
baculovirus system was increased by supplementing the medium with
silkworm hemolymph. Silkworm hemolymph increases
baculovirus-infected insect cell longevity.
[0040] Moreover, it has been shown that silkworm hemolymph inhibits
apoptosis in insect, mammalian, and human cell systems. These
results indicate that silkworm hemolymph contains a component that
inhibits apoptosis.
[0041] More recently, this anti-apoptotic fraction was isolated
from silkworm hemolymph and characterized by the present
inventors.
[0042] The fraction of silkworm hemolymph with the highest activity
was found to contain 30K proteins, which are a specific type of
plasma protein called "storage proteins" These proteins constitute
a group of structurally related proteins of approximate molecular
weight 30,000 Da. The 30K protein group consists of five proteins,
which have common characteristics in amino acid composition and
immunological activity as well as molecular weight.
[0043] The 30K protein encoded by the 30Kc6 gene of the present
invention was expressed in Escherichia coli and purified. E. coli
BL21(DE3) was used as the host for gene expression in the present
invention.
[0044] Total RNA was isolated from silkworm tissue using RNA
isolation kit, and total cDNA pool was obtained by RT-PCR using an
oligo-dT primer. The 30K protein cDNA was amplified from the cDNA
pool by PCR using specific primers. Then the amplified PCR products
were cloned into E. coli expression vector, pET-22b(+). During this
step a signal sequence contained in 30Kc6 was removed, and the
vector was designed to express the 30K protein with a 6.times. His
tag at its C-terminal. E. coli BL21(DE3) was used as the host for
gene expression.
[0045] Hereinafter, the present invention will be described in
greater detail with reference to the following examples. The
examples are given for illustration of the invention and not
intended to be limiting the present invention.
EXAMPLE 1
Plasmid containing 30K Protein cDNA Construction
[0046] The 30Kc6(GenBank Accession No.: X07552) protein cDNA was
amplified by PCR with a temperature profile of 95.degree. C. for 1
min, 56.degree. C. for 1 min, and 72.degree. C. for 1.5 min.
[0047] The forward and reverse primers were 50-AGA CAT ATG ACA CTT
GCA CCA AGA ACT-30 and 50-CAA CTC GAG GTA GGG GAC GAT GTA CCA-30,
respectively, which contain the NdeI and XhoI sites, respectively.
The forward primer contains ATG for methionine, which is necessary
for the initiation of translation in E. coli.
[0048] The amplified PCR products were cloned into a NdeI-XhoI site
in E. coli expression vector, pET-22b(+). During this step, we
removed a signal sequence contained in 30Kc6. The pET-22b(+)
carrying 30Kc6 was designed to express the 30K protein with a
6.times. His tag at its C-terminal.
EXAMPLE 2
Protein Expression, Purification, and Refolding
[0049] The pET-22b(+) carrying 30Kc6 without signal sequence, was
introduced into E. coli strain BL21(DE3) and BL21(DE3)pLysE. The
transformed bacteria were grown to OD600 of 0.5, induced with 0.5
mM isopropyl-.beta.-D-thiogalactopyranoside(IPTG), and then
incubated for 4 h. The cells were harvested by centrifugation and
resuspended in 4 ml of lysis buffer (10 mM Tris-HCl, 150 mM NaCl,
and 1 mM EDTA, pH 8.0) containing 1 mM phenylmethylsulfonyl
fluoride (PMSF) for each 100 ml of culture.
[0050] Lysozyme (0.5 mg/ml) was added and the mixture was incubated
on ice for 30 min. The suspended cells were disrupted by sonication
(Vibracell, 4 times, each for 15 sec) and centrifuged at 4.degree.
C. The precipitate containing inclusion bodies was solubilized in
6M guanidine hydrochloride overnight at 4.degree. C. This solution
was loaded on a Ni.sup.2+-charged HisTrap column (Amersham
Bioscience) and the column was washed with buffer containing 6M
urea and 20 mM imidazole several times to remove the nonspecific
binding.
[0051] Refolding of the bound protein was performed in an FPLC
(Bio-Rad, Biologic HR) using a linear urea reverse gradient (6M to
0M). The total volume and flow rate of the buffer used in the
linear gradient were 30 ml and 0.5 ml/min, respectively.
[0052] Finally, the refolded protein was eluted with elution buffer
containing 500 mM imidazole. The eluted 30K protein was desalted
into the distilled water to remove the imidazole using a HiTrap
desalting column (Amersham Bioscience) and concentrated using a
lyophilizer.
EXAMPLE 3
Quantitation of Protein
[0053] The purity of the protein obtained was determined by
scanning the 30K protein band on SDS-PAGE gel using Total Lab v
1.10(Nonlinear Dynamics). The total protein concentration was
measured using a Modified Lowery Protein Determination Kit
(Peterson's Modification of the Micro-Lowery Method; Sigma Chemical
Co.,St. Louis, Mo.).
EXAMPLE 4
Cell Culture for Anti-Apoptotic Activity Assay
[0054] Spodoptera frugiperda (Sf9) cells were cultivated in a Grace
medium(Gibco) supplemented with 10% fetal bovine serum (FBS,
Gibco), 0.35 g/L NaHCO3, and antibiotic-antimycotic(Gibco) at
28.degree. C.
[0055] HeLa cells were cultivated in DMEM(Dulbecco' smodified
Eagle's medium, Gibco) supplemented with 10% fetal bovine serum
(FBS, Gibco), Hepes, NaHCO3 (2.02 g/L), and penicillin/streptomycin
(Gibco). The cells were incubated at 37.degree. C. in humidified
air atmosphere with 5% CO2. The recombinant 30Kc6 protein expressed
in E. coli, or whole silkworm hemolymph as a control, was added to
the culture medium to investigate the effects on apoptosis.
Collection and pre-treatment of silkworm hemolymph has been
described elsewhere in detail [E. J. Kim, W. J. Rhee, T. H. Park,
Isolation and characterization of an apoptosis-inhibiting component
from the hemolymph of Bombyx mori, Biochem. Biophys. Res. Commun.
285 (2001) 224-228.]. The collected hemolymph was heat-treated at
60.degree. C. for 30 min and then chilled, and centrifuged. The
supernatant filtered with a 0.2-.mu.m membrane filter was used as a
medium supplement.
[0056] Either the baculovirus AcMNPV (Autographa californica
multiple nuclear polyhedrovirus) or actinomycin D (Sigma) was used
as an apoptosis inducer for Sf9 cells. For the baculovirus
infection, the medium was aspirated and a virus stock solution was
added.
[0057] A multiplicity of infection (MOI) of 13 was used for all the
experiments. After incubating for 1 h, the virus solution was
replaced with the medium used before the infection. Actinomycin D
dissolved in sterilized water (100 .mu.g/ml) was used to induce
apoptosis at a final concentration of 200 ng/ml in each growth
medium.
[0058] Staurosporine was used as an apoptosis inducer for HeLa
cells. Staurosporine dissolved in DMSO (300 .mu.M) was used to
induce apoptosis at a final concentration of 600 nM in each growth
medium.
EXAMPLE 5
N-terminal Amino Acid Sequencing of Recombinant 30K Proteins
[0059] SDS-PAGE was transferred to a PVDF (polyvinylidene
difluoride) membrane in transfer buffer(192 mM glycine/25 mM
Tris/20% methanol/0.037% SDS) for 90 min at 90 mA using a Bio-Rad
Trans Blot SD Semidry Transfer Cell.
[0060] After the transfer, the membrane was stained with ponceau S
(0.2% ponceau S in 1% acetic acid) and destained with deionized
water. The stained band was then cut out and air-dried. Amino acid
sequencing was carried out using the Precise Protein Sequencing
System (Applied Biosystems).
EXAMPLE 6
Apoptosis Assay
[0061] For the assay of cell viability, cell numbers were counted
under an optical microscope using a hemocytometer and viable cells
were detected using the trypan blue exclusion test. Since dead
cells absorb trypan blue (Sigma), they can be identified under an
optical microscope.
[0062] The cell viability was defined by the ratio of the viable
cell number to the total cell number. For the analysis of apoptotic
cells accompanying DNA fragmentation, cell nuclei were stained with
10 .mu.g/ml Hoechst 33258 in phosphate-buffered saline (PBS, pH7.4)
for 20 min and then observed using a fluorescence microscope
(TE300, Nikon) with a UV filter.
[0063] For the quantitative assay of apoptosis, flow cytometric
analysis was performed. Cells were collected and washed twice with
PBS (pH 7.4). The cell pellets were resuspended in cold 70% ethanol
for fixation and stored at -20.degree. C. until the FACS analysis.
The fixed cells were washed with PBS, incubated with 100 g/ml RNase
at 37.degree. C. for 1 h, and stained with 50 .mu.g/ml propidium
iodide for 15 min. A FACSCalibur flow cytometer(Becton Dickinson)
was used for this assay.
EXAMPLE 7
Culture Condition of Recombinant E. coli Containing 30K Gene
[0064] The medium consisted of 20 g of yeast extract, 10 g of
casamino acid, 0.24 g of MgSO4.7H2O, 0.01 g of CaCl2, 3 g of
KH2PO4, 2.5 g of (NH4)2HPO4, 5 g of glucose, and 200 mg of
ampicillin per liter in distilled water (pH 6.8). Seed culture was
grown in a 500 ml flask containing 80 ml of medium in a shaking
incubator at 37.degree. C., at 250 rpm for 12 h. Batch culture was
carried out in a 2.5 L jar fermentor containing 1 L of medium.
[0065] The pH was maintained at 6.8 by adding 5N HCl and 50%(v/v)
NH4OH, and the dissolved oxygen concentration was maintained above
10% air saturation by controlling the agitation speed.
Isopropylthio-.beta.-D-galactoside (IPTG) was added to the cultures
to a final concentration of 1 mM, and culture continued for 20
h.
EXAMPLE 8
Preparation of Recombinant 30Kc12 Protein
[0066] The pET-22b(+) carrying 30Kc12 (GenBank Accession No.:
X07553), instead of the pET-22b(+)carrying 30Kc6 of Example 2, is
introduced into E. coli strain BL21(DE3) and BL21(DE3)pLysE. The
transformed bacteria thus prepared, are treated by the process
described in Example 2 to prepare the recombinant anti-apoptotic
protein 30Kc12.
EXAMPLE 9
Preparation of Recombinant 30Kc19 Protein
[0067] The pET-22b(+) carrying 30Kc19(GenBank Accession No.:
X07554), instead of the pET-22b(+)carrying 30Kc6 of Example 2 is
introduced into E. coli strain BL21(DE3) and BL21(DE3)pLysE. The
transformed bacteria thus prepared, are treated by the process
described in Example 2 to prepare the recombinant anti-apoptotic
protein 30Kc19.
EXAMPLE 10
Preparation of Recombinant 30Kc21 Protein
[0068] The pET-22b(+) carrying 30Kc21(GenBank Accession No.:
X07555), instead of the pET-22b(+)carrying 30Kc6 of Example 2, is
introduced into E.coli strain BL21(DE3) and BL21(DE3)pLysE. The
transformed bacteria thus prepared, are treated by the process
described in Example 2 to prepare the recombinant anti-apoptotic
protein 30Kc21.
EXAMPLE 11
Preparation of Recombinant 30Kc23 Protein
[0069] The pET-22b(+) carrying 30Kc23(GenBank Accession No.:
X07556), instead of the pET-22b(+)carrying 30Kc6 of Example 2, is
introduced into E.coli strain BL21(DE3) and BL21(DE3)pLysE. The
transformed bacteria thus prepared, are treated by the process
described in Example 2 to prepare the recombinant anti-apoptotic
protein 30Kc23.
[0070] While the present invention has been described with
reference to particular embodiment thereof, there can be various
modifications on the basis of the spirit of the present invention.
Sequence CWU 1
1
10 1 771 DNA Bombyx mori 1 atgagactga ctttgtttgc cttcgtcctc
gccgtgtgtg cgctggcttc taacgccaca 60 cttgcaccaa gaactgatga
cgtactggca gagcagctgt atatgagtgt cgtcattggt 120 gaatacgaga
ccgctatcgc caaatgctct gaatatctga aggaaaagaa gggagaggtt 180
atcaaggaag ccgtgaagcg tctgatcgaa aacggcaaga ggaacaccat ggacttcgcc
240 taccagttat ggacaaagga tggaaaggaa atcgtcaaat cttacttccc
catccagttt 300 agagtgatct tcaccgagca gactgtcaag ctcataaaca
aaagggacca tcacgccctc 360 aagttgatcg accaacaaaa ccacaacaaa
attgcattcg gtgactccaa agacaaaacc 420 agcaagaaag tctcctggaa
gtttaccccc gtgttggaaa acaacagagt atacttcaag 480 atcatgtcca
ccgaagacaa acagtacctg aagctcgata acacgaaagg ttctagtgat 540
gaccgtatca tctacggtga tagcaccgct gacaccttca aacaccactg gtaccttgag
600 ccctccatgt acgaaagcga cgtcatgttc ttcgtctaca accgagagta
caacagtgtt 660 atgacacttg atgaagatat ggccgccaac gaagaccgtg
aagccttggg gcacagcgga 720 gaagtttccg gttatcccca actttttgca
tggtacatcg tcccctacta a 771 2 795 DNA Bombyx mori 2 atgaaacttc
tcgttgtgtt cgcaatgtgc gtgcctgccg ccagcgccgg cgtcgtggaa 60
ctatccgcgg acagcatgag cccttctaac caagacctcg aggacaaact gtacaacagc
120 atcctcaccg gtgactacga cagtgctgta cgtaagagct tggaatacga
gagccaaggc 180 cagggcagca tcgttcaaaa tgtagttaac aatctgatta
ttgacaagag acggaacacc 240 atggagtact gctacaagct gtgggtcggc
aacggacagg atattgtcaa aaagtacttc 300 ccattaagct ttagactcat
catggccgga aactacgtca agctcattta cagaaactac 360 aacctcgctc
tgaagctcgg ttccacaacc aatccctcga atgagagaat tgcctacggc 420
gatggtgttg acaagcacac tgacctcgtc agttggaagt tcattacctt gtgggagaac
480 aacagagtgt acttcaaggc ccacaacact aagtacaacc agtacttgaa
gatgagtacg 540 tcgacttgca actgcaacgc tcgggaccgt gttgtatacg
gcggcaacag cgctgacagc 600 accagggagc aatggttctt ccagcccgcc
aagtacgaaa acgacgtcct gttcttcatc 660 tacaatcgcc aattcaacga
tgccttggag ctcggtacga tcgtgaacgc ctcgggagac 720 cgcaaggccg
ttggacacga tggtgaagtc gccggtcttc ctgacatcta ctcgtggttc 780
attacacctt tctaa 795 3 765 DNA Bombyx mori 3 atgaagcccg ctatagttat
tctatgtctt ttcgtggcat ctctgtatgc tgcagattcc 60 gacgtcccta
acgacattct ggaggagcag ctttacaata gcgtcgtcgt cgccgattac 120
gacagtgcgg ttgaaaagag caagcattta tacgaggaga agaagagcga agtcatcaca
180 aatgtcgtga acaaactgat acgaaacaac aagatgaact gcatggagta
cgcctatcaa 240 ctttggctcc agggctccaa ggacatcgtc cgggattgtt
tcccagttga gttcagactt 300 atcttcgccg aaaacgcgat taagcttatg
tacaagcgcg acggtctcgc tttgacgctg 360 agcaatgatg ttcaaggcga
cgatggcaga cctgcctacg gcaaggacaa gacaagcccg 420 agagtcagct
ggaagttaat cgctctgtgg gagaacaaca aggtctactt caagatcttg 480
aacactgaac gtaaccaata cttggtattg ggagtcggca ctaactggaa cggcgaccat
540 atggccttcg gagtcaacag cgtcgatagt ttcagagccc agtggtacct
gcagcctgct 600 aagtacgaca atgacgtctt gttctacatc tacaaccgtg
aatacagcaa ggctttgaca 660 ctgtcgagga cggttgagcc ctcgggtcac
cgcatggcct ggggatacaa cggcagagta 720 atcggaagtc ccgaacatta
cgcttggggt ataaaggctt tctaa 765 4 756 DNA Bombyx mori 4 atgaaatttg
tcgtggtgtt cgcgtcgtgc gtgctcgccg tcagcgccgg cgtcactgaa 60
atgtccgcgg caagcatgag cagttctaac aaagaactcg aggagaaact gtacaacagc
120 atcctcaccg gtgactacga cagcgctgtc cgtcagagct tggaatacga
gaaccaaggc 180 aagggctcca tcatccagaa tgtagttaac aacctgatca
ttgacggaag tcggaacacc 240 atggagtact gctacaagct gtgggtcggc
aacggacagc acattgtcag aaagtacttc 300 ccctataact ttagactcat
catggccgga aacttcgtta agctcattta cagaaactac 360 aacctcgctc
tgaagctcgg ccccactctt gatcccgcga acgagagact tgcatacggc 420
gatggtaagg aaaagaacag cgacctcatc agttggaagt ctcattacct tgtgggagaa
480 caacatagtg tacttcaaga tccaccaaca ctaagctaca accagtactt
gaaactaagt 540 tcgactactg actgcaacac tcaagaccgt attatattcg
gcaccaacac cgccgacacc 600 accagggagc agtggttcct ccagcccacg
aagtacgaaa acgacgtcct gttcttcatc 660 tacaatcgcg aggtacaacg
agtggctttg aagctcggta ggattgtgga cgcttcggga 720 gaccgtagtg
gcatttggac acgatggatg aagtag 756 5 795 DNA Bombyx mori 5 atgaaatttc
tcgtggtgtt cgcggtcgtg cgtgcctgcg tcacgccggc gtgcgctgaa 60
atgtccgcgg taagcatgag cagttctaac aaagaactcg aggagaaact gtacaacagc
120 atcctcaccg gcgactacga cagtgctgtc cgccagagct tggaatacga
gagccaaggc 180 aagggctcca tcatccagaa tgtagttaac aacctgatca
ttgacaagag acggaacacc 240 atggagtact gctacaagct gtgggtcggc
aacggacagg aaattgttag aaagtacttc 300 ccattaaact ttagactcat
catggccgga aactatgtca agatcattta cagaaactac 360 aacctcgctc
tgaagctcgg ttccacaacc aatccctcga atgagagaat tgcctacggc 420
gatggtgtag acaagcatac tgaactcgtc agttggaagt tcattacctt gtgggagaac
480 aacagagtgt acttcaagat ccacaacact aagtacaacc agtacttgaa
gatgagtacg 540 acgacttgca actgcaacag tcgcgaccgt gttgtatacg
gcggcaacag cgctgacagc 600 accagggagc aatggttctt ccagcccgcc
aagtacgaaa acgacgtcct gttcttcatc 660 tacaatcgcc aattcaacga
tgccttggag ctcggtacga tcgtgaacgc ctcgggagac 720 cgcaaggccg
ttggacacga tggtgaagtc gccggtcttc ctgacatcta ctcgtggttc 780
attacacctt tctaa 795 6 238 PRT Bombyx mori 6 Met Thr Leu Ala Pro
Arg Thr Asp Asp Val Leu Ala Glu Gln Leu Tyr 1 5 10 15 Met Ser Val
Val Ile Gly Glu Tyr Glu Thr Ala Ile Ala Lys Cys Ser 20 25 30 Glu
Tyr Leu Lys Glu Lys Lys Gly Glu Val Ile Lys Glu Ala Val Lys 35 40
45 Arg Leu Ile Glu Asn Gly Lys Arg Asn Thr Met Asp Phe Ala Tyr Gln
50 55 60 Leu Trp Thr Lys Asp Gly Lys Glu Ile Val Lys Ser Tyr Phe
Pro Ile 65 70 75 80 Gln Phe Arg Val Ile Phe Thr Glu Gln Thr Val Lys
Leu Ile Asn Lys 85 90 95 Arg Asp His His Ala Leu Lys Leu Ile Asp
Gln Gln Asn His Asn Lys 100 105 110 Ile Ala Phe Gly Asp Ser Lys Asp
Lys Thr Ser Lys Lys Val Ser Trp 115 120 125 Lys Phe Thr Pro Val Leu
Glu Asn Asn Arg Val Tyr Phe Lys Ile Met 130 135 140 Ser Thr Glu Asp
Lys Gln Tyr Leu Lys Leu Asp Asn Thr Lys Gly Ser 145 150 155 160 Ser
Asp Asp Arg Ile Ile Tyr Gly Asp Ser Thr Ala Asp Thr Phe Lys 165 170
175 His His Trp Tyr Leu Glu Pro Ser Met Tyr Glu Ser Asp Val Met Phe
180 185 190 Phe Val Tyr Asn Arg Glu Tyr Asn Ser Val Met Thr Leu Asp
Glu Asp 195 200 205 Met Ala Ala Asn Glu Asp Arg Glu Ala Leu Gly His
Ser Gly Glu Val 210 215 220 Ser Gly Tyr Pro Gln Leu Phe Ala Trp Tyr
Ile Val Pro Tyr 225 230 235 7 249 PRT Bombyx mori 7 Met Gly Val Val
Glu Leu Ser Ala Asp Ser Met Ser Pro Ser Asn Gln 1 5 10 15 Asp Leu
Glu Asp Lys Leu Tyr Asn Ser Ile Leu Thr Gly Asp Tyr Asp 20 25 30
Ser Ala Val Arg Lys Ser Leu Glu Tyr Glu Ser Gln Gly Gln Gly Ser 35
40 45 Ile Val Gln Asn Val Val Asn Asn Leu Ile Ile Asp Lys Arg Arg
Asn 50 55 60 Thr Met Glu Tyr Cys Tyr Lys Leu Trp Val Gly Asn Gly
Gln Asp Ile 65 70 75 80 Val Lys Lys Tyr Phe Pro Leu Ser Phe Arg Leu
Ile Met Ala Gly Asn 85 90 95 Tyr Val Lys Leu Ile Tyr Arg Asn Tyr
Asn Leu Ala Leu Lys Leu Gly 100 105 110 Ser Thr Thr Asn Pro Ser Asn
Glu Arg Ile Ala Tyr Gly Asp Gly Val 115 120 125 Asp Lys His Thr Asp
Leu Val Ser Trp Lys Phe Ile Thr Leu Trp Glu 130 135 140 Asn Asn Arg
Val Tyr Phe Lys Ala His Asn Thr Lys Tyr Asn Gln Tyr 145 150 155 160
Leu Lys Met Ser Thr Ser Thr Cys Asn Cys Asn Ala Arg Asp Arg Val 165
170 175 Val Tyr Gly Gly Asn Ser Ala Asp Ser Thr Arg Glu Gln Trp Phe
Phe 180 185 190 Gln Pro Ala Lys Tyr Glu Asn Asp Val Leu Phe Phe Ile
Tyr Asn Arg 195 200 205 Gln Phe Asn Asp Ala Leu Glu Leu Gly Thr Ile
Val Asn Ala Ser Gly 210 215 220 Asp Arg Lys Ala Val Gly His Asp Gly
Glu Val Ala Gly Leu Pro Asp 225 230 235 240 Ile Tyr Ser Trp Phe Ile
Thr Pro Phe 245 8 238 PRT Bombyx mori 8 Met Ala Asp Ser Asp Val Pro
Asn Asp Ile Leu Glu Glu Gln Leu Tyr 1 5 10 15 Asn Ser Val Val Val
Ala Asp Tyr Asp Ser Ala Val Glu Lys Ser Lys 20 25 30 His Leu Tyr
Glu Glu Lys Lys Ser Glu Val Ile Thr Asn Val Val Asn 35 40 45 Lys
Leu Ile Arg Asn Asn Lys Met Asn Cys Met Glu Tyr Ala Tyr Gln 50 55
60 Leu Trp Leu Gln Gly Ser Lys Asp Ile Val Arg Asp Cys Phe Pro Val
65 70 75 80 Glu Phe Arg Leu Ile Phe Ala Glu Asn Ala Ile Lys Leu Met
Tyr Lys 85 90 95 Arg Asp Gly Leu Ala Leu Thr Leu Ser Asn Asp Val
Gln Gly Asp Asp 100 105 110 Gly Arg Pro Ala Tyr Gly Lys Asp Lys Thr
Ser Pro Arg Val Ser Trp 115 120 125 Lys Leu Ile Ala Leu Trp Glu Asn
Asn Lys Val Tyr Phe Lys Ile Leu 130 135 140 Asn Thr Glu Arg Asn Gln
Tyr Leu Val Leu Gly Val Gly Thr Asn Trp 145 150 155 160 Asn Gly Asp
His Met Ala Phe Gly Val Asn Ser Val Asp Ser Phe Arg 165 170 175 Ala
Gln Trp Tyr Leu Gln Pro Ala Lys Tyr Asp Asn Asp Val Leu Phe 180 185
190 Tyr Ile Tyr Asn Arg Glu Tyr Ser Lys Ala Leu Thr Leu Ser Arg Thr
195 200 205 Val Glu Pro Ser Gly His Arg Met Ala Trp Gly Tyr Asn Gly
Arg Val 210 215 220 Ile Gly Ser Pro Glu His Tyr Ala Trp Gly Ile Lys
Ala Phe 225 230 235 9 236 PRT Bombyx mori 9 Met Gly Val Thr Glu Met
Ser Ala Ala Ser Met Ser Ser Ser Asn Lys 1 5 10 15 Glu Leu Glu Glu
Lys Leu Tyr Asn Ser Ile Leu Thr Gly Asp Tyr Asp 20 25 30 Ser Ala
Val Arg Gln Ser Leu Glu Tyr Glu Asn Gln Gly Lys Gly Ser 35 40 45
Ile Ile Gln Asn Val Val Asn Asn Leu Ile Ile Asp Gly Ser Arg Asn 50
55 60 Thr Met Glu Tyr Cys Tyr Lys Leu Trp Val Gly Asn Gly Gln His
Ile 65 70 75 80 Val Arg Lys Tyr Phe Pro Tyr Asn Phe Arg Leu Ile Met
Ala Gly Asn 85 90 95 Phe Val Lys Leu Ile Tyr Arg Asn Tyr Asn Leu
Ala Leu Lys Leu Gly 100 105 110 Pro Thr Leu Asp Pro Ala Asn Glu Arg
Leu Ala Tyr Gly Asp Gly Lys 115 120 125 Glu Lys Asn Ser Asp Leu Ile
Ser Trp Lys Ser His Tyr Leu Val Gly 130 135 140 Glu Gln His Ser Val
Leu Gln Asp Pro Pro Thr Leu Ser Tyr Asn Gln 145 150 155 160 Tyr Leu
Lys Leu Ser Ser Thr Thr Asp Cys Asn Thr Gln Asp Arg Ile 165 170 175
Ile Phe Gly Thr Asn Thr Ala Asp Thr Thr Arg Glu Gln Trp Phe Leu 180
185 190 Gln Pro Thr Lys Tyr Glu Asn Asp Val Leu Phe Phe Ile Tyr Asn
Arg 195 200 205 Glu Val Gln Arg Val Ala Leu Lys Leu Gly Arg Ile Val
Asp Ala Ser 210 215 220 Gly Asp Arg Ser Gly Ile Trp Thr Arg Trp Met
Lys 225 230 235 10 242 PRT Bombyx mori 10 Met Val Ser Met Ser Ser
Ser Asn Lys Glu Leu Glu Glu Lys Leu Tyr 1 5 10 15 Asn Ser Ile Leu
Thr Gly Asp Tyr Asp Ser Ala Val Arg Gln Ser Leu 20 25 30 Glu Tyr
Glu Ser Gln Gly Lys Gly Ser Ile Ile Gln Asn Val Val Asn 35 40 45
Asn Leu Ile Ile Asp Lys Arg Arg Asn Thr Met Glu Tyr Cys Tyr Lys 50
55 60 Leu Trp Val Gly Asn Gly Gln Glu Ile Val Arg Lys Tyr Phe Pro
Leu 65 70 75 80 Asn Phe Arg Leu Ile Met Ala Gly Asn Tyr Val Lys Ile
Ile Tyr Arg 85 90 95 Asn Tyr Asn Leu Ala Leu Lys Leu Gly Ser Thr
Thr Asn Pro Ser Asn 100 105 110 Glu Arg Ile Ala Tyr Gly Asp Gly Val
Asp Lys His Thr Glu Leu Val 115 120 125 Ser Trp Lys Phe Ile Thr Leu
Trp Glu Asn Asn Arg Val Tyr Phe Lys 130 135 140 Ile His Asn Thr Lys
Tyr Asn Gln Tyr Leu Lys Met Ser Thr Thr Thr 145 150 155 160 Cys Asn
Cys Asn Ser Arg Asp Arg Val Val Tyr Gly Gly Asn Ser Ala 165 170 175
Asp Ser Thr Arg Glu Gln Trp Phe Phe Gln Pro Ala Lys Tyr Glu Asn 180
185 190 Asp Val Leu Phe Phe Ile Tyr Asn Arg Gln Phe Asn Asp Ala Leu
Glu 195 200 205 Leu Gly Thr Ile Val Asn Ala Ser Gly Asp Arg Lys Ala
Val Gly His 210 215 220 Asp Gly Glu Val Ala Gly Leu Pro Asp Ile Tyr
Ser Trp Phe Ile Thr 225 230 235 240 Pro Phe
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